Methods for extracting bioactive small rnas from plants and mushrooms

ABSTRACT

The present invention refers to a method for isolating a fraction enriched of small RNA molecules from a fungal or plant sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/770,008, filed Apr. 20, 2018, which is a 371 of PCT/EP2016/076050,filed Oct. 28, 2016, which in turn claims the benefit of European PatentApplication No. 15191884.4, filed Oct. 28, 2015, contents of each ofwhich are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to methods for isolating or extractingsmall RNA (sRNA) molecules from a fungal or plant sample. Said sRNAscomprise single stranded RNAs and/or single stranded RNAs with partialalignment but more preferably double stranded RNA with or withoutperfect sequence complementarity. The obtained sRNA may be used in foodpreparations and/or in the nutraceutical, cosmeceutical orpharmaceutical fields.

BACKGROUND OF THE INVENTION

WO2005012523 refers to the use of methods and compositions for theisolation of small RNA molecules (100 nucleotides or fewer), such asmicroRNA and siRNA molecules. WO2011086195 refers to a method forisolating small RNA from a sample comprising binding the RNA to silicaparticles by contacting the sample with a) at least one alcohol, b) atleast one chaotropic salt comprising a chaotropic anion selected fromthe group consisting of trichloroacetate, perchlorate andtrifluoroacetate and c) silica particles and separating the bound RNAfrom the rest of the sample. Compositions and kits are also provided toefficiently isolate small RNA molecules from samples, in particularbiological samples such as blood, blood products tissue and body fluids.WO2014033326 refers to a method for isolating RNA including small RNAhaving a size of 200nt or less from a sample, comprising the followingsteps: a) providing a composition comprising RNA and a chaotropic agent;b) adding alcohol; c) incubating the mixture for at least 2 min; d)adding additional alcohol to the mixture to adjust the overall alcoholconcentration in the mixture to>=50%; e) binding RNA contained in themixture to a nucleic acid binding solid phase; f) optionally washing thebound RNA; g) optionally eluting RNA from the solid phase.

Due to the step-wise addition of alcohol, the overall RNA yield and theyield of small RNA is improved.

WO2007100934 refers to extraction compositions and methods for the rapidand efficient isolation of small RNA molecules from a biological sample.In particular, the extraction compositions, when contacted with abiological sample, releases the small RNA molecules from the othermolecules in a biological sample, and the released small RNA moleculesmay then be isolated.

WO2010033652 refers to a method for the extraction and purification ofsmall RNA from a sample solution. Accordingly, a sample is first mixedwith an organic solvent to form a mixture containing the solvent. Themixture is applied to a first mineral support for large RNA to bind. Thefiltrate is collected which contain unbound small RNA, and is mixed witha second organic solvent to form a second mixture containing the secondsolvent. This second mixture is applied to a second mineral support forsmall RNA to bind. After a wash step, the small RNA is eluted. Alsoprovided is a method for the isolation of large RNA, by eluting thelarge RNA from the first mineral support. In addition, total protein ispresent in the filtrate and can be isolated by a conventional method.

WO9506652 refers to compositions and methods for isolating nucleic acidswith lengths greater than about 50 bases, from cells, gels, solutionsand other media, in which nucleic acids occur in vivo or in vitro. Thecompositions of the invention are mixtures of the silica materialssilica gel and glass particles, particularly glass microfibers; suchmixtures combined with chaotropic salts, such as guanidinium chloride orguanidinium thiocyanate; and suspensions of such mixtures in aqueoussolutions of chaotropic salts. In the methods of the invention, anaqueous solution comprising nucleic acid is mixed with an aqueoussolution of chaotropic salts and the resulting solution is contactedwith a mixture of the silica materials, whereupon the nucleic acid inthe solution binds to the silica materials. The chaotropic salts andcomponents, other than the nucleic acid adsorbed to the silicamaterials, from the aqueous solution treated by the method of theinvention are washed from the silica materials. Finally, the nucleicacid can be obtained by elution from the silica materials. The methodsprovide nucleic acid in water or buffer, such as TE buffer, free ofcontamination by any salt or macromolecule that would interfere withfurther processing or analysis.

WO2007103485 relates to methods, kits, and compositions for purifyingsmall RNA molecules. In particular, the present invention providesmethods for purifying small RNA molecules from a sample containing bothsmall RNA molecules and larger RNA molecules using a compaction agentand a RNA binding matrix, as well as compositions and kits forpracticing such methods. In certain embodiments, the compaction agentcomprises a plurality of metal-amine-halide molecules.

All known methods for isolation of small RNA from plants or mushroomshave been developed mainly for analytical scopes and are not suitablefor efficient and isolation of safe small RNA molecules from fungiand/or plants which may be used in food industry. Thus they generallyare employed with small amounts of tissues (mg or grams) and aim atproviding the full spectrum of RNA species present in the tissue. Thisinvolves the use of chemical and physical strategies to preventRNAse-mediated degradation of some types or RNAs most significantlymRNAs and other ssRNAs. The strategies used to prevent RNAse activity inthe sample include the use of toxic solvents agents (i.e. phenol),solvents (chloroform) or chaotropic agents (TFA, PCA and guanidine HCl).All these methods are cumbersome and not amenable for scale-up orindustrialization through the need for phase separation, centrifugationand/or the use (and disposal) of expensive or toxic solvents orreagents. In view of the non-sequence dependent immunomodulant activityof sRNA species recently demonstrated Plant microrna as novelimmunomodulatory agents. Sci. Rep. 2016, 6:25761, Cavalieri et al.),there is a need to perfect a method for extraction and isolation ofsRNAs from high quantities plant or fungi tissues and which is designedto obtain maximum extraction particularly of the dsRNA species (20-60 bplength) which are responsible for the immunomodulant activity of sRNAextracts (Plant microrna as novel immunomodulatory agents. Sci. Rep.2016, 6:25761, Cavalieri et al.). Moreover, such method should notinvolve the use of toxic or costly solvents and ideally do not containcomplex technical steps (i.e. high speed centrifugation) that render itunsuitable or too expensive for scale-up.

SUMMARY OF THE INVENTION

The method for isolating a fraction enriched of small RNA (sRNA)molecules from a fungal or plant sample object of the present inventionsuccessfully allows to obtain over 100 mg of sRNA from a Kg of plant ormushroom tissue. The sRNA extract can then be further treated withRNAses to selectively remove ssRNA species and treated with a proteincontaining dsRNA binding domain which selectively recognizes dsRNA ofbetween 21 to 24 bp to selectively purify the dsRNA species of 21 and 24base pairs. The final extract can be safely used as a dietary supplementor for the nutraceuticals, pharmaceutical or cosmeceuticals industry.

It is therefore an object of the invention a method for isolating afraction enriched of small RNA molecules from a fungal and/or plantsample comprising:

a) adding a lysis buffer to a powder or homogenized sample of fungaland/or plant tissue or cells sample to obtain a lysate; or

a′) incubating fungal and/or plant tissue or cells with a bicarbonatesolution at a temperature of 50-100° C., separate liquid from solidphase to obtain a liquid phase and

b) adding an alcohol solution to the lysate obtained is step a) or tothe liquid phase obtained in step a′) to obtain a solution;

c) loading the obtained solution to a solid support able to bind smallRNA molecules;

d) eluting small RNA molecules from said solid support.

Said homogenized or powder sample is preferably obtained by homogenizingfungal and/or plant tissue or cells to obtain a powder or an homogenizedsample or is a lyophilized powder.

Before homogenizing fungal and/or plant tissue or cells, the fungi orplant tissue or cells are preferably snap frozen or freeze dried.

In the method according to the invention, the ratio w/v between powderand lysis buffer in step

a) is preferably of 1:1 to 1:4, preferably of 1:2.

Preferably, in the method according to the invention, the lysis buffercomprises:

a) 10-100 mM of Tris-HC1 at a pH of 5-10, preferably 50 mM of Tris-HClat a pH of 8-8.5,

b) 100-500 mM, preferably 100-300 mM, more preferably 200 mM, of NaCl,

c) EDTA 5-50 mM, preferably EDTA 10-20 mM, more preferably EDTA 10 mM,

d) SDS 0.5-10% (w/v), preferably SDS 2-4% (w/v), more preferably SDS 2%(w/v).

In a preferred embodiment the lysis buffer comprises: 50 mM Tris-HCl (pH8.5), 200 mM NaCl, 10 mM EDTA and 2% SDS (w/v).

The bicarbonate solution is a preferably a diluted sodium bicarbonatesolution, more preferably a 5-100 mM NaHCO₃ solution, even morepreferably a 30 mM NaHCO₃. Other bicarbonate solutions may be alkalimetal bicarbonate, preferably selected from the group consisting ofsodium bicarbonate, potassium bicarbonate, and mixtures thereof.

The method of the invention may further comprise after step a′), a stepa″) wherein the liquid phase is discarded and a diluted bicarbonatesolution is added at a temperature of 60-100° C. and wherein the dilutedbicarbonate solution is a 5-10 mM NaHCO₃, solution, preferably a 8.75 mMNaHCO₃ solution, said step a″) being optionally repeated.

The liquid may be separated from the solid phase and then be subjectedto the addition of isopropanol.

The temperature of the diluted bicarbonate is preferably of about 95,100 or 60° C.

Before step a′) fungal and/or plant tissue or cells is preferablyincubated with a diluted bicarbonate solution at 0-10° C., preferably at4° C., wherein the diluted bicarbonate solution is a 5-10 mM NaHCO₃,solution, preferably a 8.75 mM NaHCO₃, solution. The incubation may be10-20 h long, preferably 16 h. The liquid phase is then discarded. Thestarting plant or mushroom tissue may be intact or coarsely chopped.

In the method according to the invention the alcohol is preferablyisopropanol, ethanol or any alcohol able to reduce the activity of waterand therefore the solvation of sRNAs and promote their binding to thesolid support.

More preferably, the alcohol solution comprises isopropanol at a finalconcentration of 60% v/v.

Preferably, the small RNA molecules are eluted from the solid supportwith RNAse-free water.

In a preferred embodiment of the invention, the small RNA molecules areeluted from the solid support at a temperature of about 50° C. to about100° C., preferably at 55 or 65° C.

Preferably, the solid support is a mineral support or polymer support.

More preferably the mineral support or polymer support is a columncomprising silica or silica gel or silicon dioxide particles.

More preferably the mineral or polymer support is a set of beads made ofan absorptive polymer, preferably silica beads.

The set of beads are preferably collected by centrifugation, filtration,or magnetic capture.

The method according to the invention may further comprise capturing theeluted small RNA molecules. In some embodiments e.g. the sample issubsequently filtered after passage through a capture structure. Thecapture step can include filtration using a pressure-driven system orgravity-based system (for example, centrifugation).

In another embodiment, the method further comprises:

step e) selectively removing ssRNAs with RNAse treatment from the elutedsmall RNA molecules

and/or

step f) treating the eluted small RNA molecules with an agent whichbinds with high affinity to siRNA duplexes of selectively 21-25 nt, toenrich the 21-24 bp dsRNA fraction. Preferably said agent is p19.

In step e) it may be used RNAse A or any RNAse able to completelydegradate ssRNA.

Preferably, the small RNA molecules (and the fraction enriched of smallRNA molecules) include miRNA, siRNA, snRNA, snoRNA, and/or tRNAmolecules, more preferably the small RNA molecules consist of at most100 nucleotides, preferably at most 70 nucleotides, or more preferablyat most 30 nucleotides.

The small RNA molecules preferably consist of between 21 and 24nucleotide, more preferably between 19 and 24 nucleotides.

Said small RNA molecules are preferably in the single stranded and/ordouble stranded configurations.

Said small RNA molecules are preferably characterized by the presence ofa phosphate group at the 5′ ends and/or a methyl group at the 3′ ends.The small RNA molecules are preferably miRNA, mature miRNA and/or siRNAmolecules.

Another object of the invention is small RNA molecules obtained by themethod as above defined.

Further objects of the invention are a food and/or food additive and/ordietary supplement and/or a nutraceutical product comprising the smallRNA molecules obtainable with the method as above defined, a method forproducing a second food and/or a nutraceutical product comprising theaddition to a first food and/or nutraceutical product of the small RNAmolecules obtained by above method of the invention.

Another object of the invention is a pharmaceutical or cosmeticcomposition comprising the small RNA molecules obtainable with themethod as above defined.

The pharmaceutical or cosmetic composition as above defined, the foodand/or food additive and/or dietary supplement and/or a nutraceuticalproduct as above defined, preferably comprise:

a) lipids, more preferably at least one liposome, b) an exosome, c) apolymeric nanoparticle, more preferably a chitosan-based particle, or d)a β1,3-D-glucan particle.

The above method according to the invention is herein also defined as“MRG extraction method”.

Therefore, the present method allows to obtain a fraction enriched ofsmall RNA (sRNA) molecules.

DETAILED DESCRIPTIONS

In the context of the present invention sRNAs comprise double strandedand/or single stranded RNAs and/or single stranded RNAs with partialalignment.

In the context of the present invention “fraction” means e.g. a sample,an extract, an eluted.

In the context of the present invention the term “fungi” or “fungal”comprises the phyla Ascomycota, Basidiomycota, Chytridiomycota, andZygomycota as well as the Oomycota and all mitosporic fungi (as definedby Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi,8th edition, 1995, CAB International, University Press, Cambridge, UK).The fungal cell(s) can be any fungal cell, meaning any cell presentwithin or derived from an organism belonging to the Kingdom Fungi. Themethods of the invention are applicable to all fungi and fungal cells.In one embodiment of the invention, the fungus may be a mould, or moreparticularly a filamentous fungus. In other embodiments of theinvention, the fungus may be a yeast (e.g. Saccharomices or Pichia), orAspergillus, Rhizopus, Neurospora. In one embodiment the fungus may bean ascomycetes fungus, i.e. a fungus belonging to the Phylum Ascomycota.

In preferred, but non-limiting, embodiments of the invention the fungusis chosen from the group consisting of edible fungi: Agaricus bisporus,and fungi of the genuses Agrocybe, Amanita, Armillaria, Artomyces,Astraeus, Aureoboletus, Auricularia, Boletus, Bovista, Butyriboletus,Calbovista, Calocybe, Calvatia, Candy Cap, Cantharellula, Cantharellus,Chalciporus, Chanterelle, Chroogomphus, Clavaria, Clavariadelphus,Clavulina, Clitocybe, Clitopilus, Coprinellus, Coprinopsis, Coprinus,Corn smut, Cortinarius, Craterellus, Cyanoboletus, Cystoderma,Cystodermella, Dacryopinax, Disciotis, Entoloma, Eritadenine,Exsudoporus, Fistulina, Floccularia, Geopora, Gliophorus, Gomphidius,Gomphus, Goossensia, Grifola, Guepinia, Gymnopus, Gyromitra, Gyroporus,Handkea, Harrya, Helvella, Hemileccinum, Hericium, Hydnum, Hygrocybe,Hygrophorus, Hypomyces, Hypsizygus, Imleria, Infundibulicybe, Laccaria,Laccocephalum, Lactarius, Lactifluus, Laetiporus, Lanmaoa, Leccinellum,Leccinum, Lentinula, Lepista, Leucopholiota, Lobaria, Lycoperdon,Mackintoshia, Marasmius, Melanoleuca, Meripilus, Morchella, Mycenastrum,Penicillium, Phallus, Phylloporus, Pleurocybella, Pleurotus, Pluteus,Polyozellus, Psathyrella, Pseudohydnum, Ramaria, Ramariopsis,Rhizopogon, Rhodocybe, Russula, Saccharomyces, Sarcodon, Sarcosphaera,Sparassis, Strobilurus, Stropharia, Suillellus, Suillus, Termitomyces,Tremella, Tricholoma, Tylopilus Verpa, Volvariella, Volvopluteus,Xerocomellus, Xerocomus, Xeromphalina.

In the context of the present invention the term “fungi” or “mushroom”are interchangeable terms.

The term “fungal cell” encompasses fungal cells of all types and at allstages of development, including specialised reproductive cells such assexual and asexual spores. As used herein the fungal cell encompassesthe fungus as such and also other life forms of the fungus, such ashaustoria, conidia, mycelium, penetration peg, spore, zoospores etc.

In the context of the present invention, the term “plant” encompassesany member of the plant-kingdom according to the Linnaeus definition.Examples of plants are plants included in the genera Arabidopsis, ase.g. Arabidopsis thaliana, or Phaseolus, as e.g. Phaseolus vulgaris, orNicotiana, as e.g. Nicotiana tabacum, or Glycine, as e.g. Glycine max,or Gossypium, as e.g. Gossypium arboreum, or Brassica, as e.g. Brassicanapus, or Vitis, as e.g. Vitis vinifera, or Beta, as e.g. Beta vulgaris,or Triticum, as e.g. Triticum aestivum, or Solanum, as e.g. Solanumlycopersicum, Solanum tuberosum and Solanum melongena L., or Musa, ase.g. Musa acuminata and Musa balbisiana, or Fragaria, as e.g. Fragariavesca, Fragaria viridis and Fragaria moschata, or Oryza, as e.g. Oryzasativa, or Hordeum, as e.g. Hordeum vulgare, or Olea, as e.g. Oleaeuropaea, or Malus, as e.g. Malus domestica, or Allium, e.g. Alluimcepa, or Pisum, e.g. Pisum sativum, or Mentha, or Cuminum, e.g. Cuminumcyminum. The term plant also comprises lychens and algae and fruitingsand any part of the plant.

The fungal or plant sample may be homogenized or fractionated, forexample by grinding it into a fine powder. The expert in the art willknow what is intended for fine powder.

The plant sample may comprise cotyledons, leaves (fresh or dry),sprouts, root, flowers, bulb, seed and/or fruits.

In the present invention, the fraction enriched of small RNA moleculesmay comprise sRNA and other RNA molecules.

The powder is mixed with a lysis buffer (LB) to release the sRNA fromthe fungal or plant cells.

The LB is preferably preheated at a temperature of 30-70° C., morepreferably at about 55° C.

The lysis buffer may comprise a Buffer Tris HC1 or any other properbuffer, having a concentration range of 10-100 mM and pH range of 7-9.5.Preferably the pH of the LB is of 8.5.

The lysis buffer may comprise NaCl or any other proper chaotropic agent,in a concentration range of 100-500 mM, preferably of 0.2 M.

The lysis buffer may comprise EDTA or any other proper chelating agent,in a concentration range of 5-50 mM, preferably of 10 or 20 mM.

The lysis buffer may comprise SDS or any other proper anionicsurfactants, in a concentration range of 0.5%-10%, preferably of 2%.

In another embodiment, the lysis buffer may be a 0.3M solution of sodiumbicarbonate. When fresh tissue is used (grinded in Liquid nitrogen), theratio between the weight of the powder (FW (Fresh Weight)) and LB may bein the range of 1:1 to 1:5. In the case of lyophilized tissue (which isthen grinded), the ratio between DW (Dry Weight) and LB may be in therange of 1:5 to 1:25.

The incubation with LB may be carried out at a temperature of 20-60° C.,preferably at RT. The incubation is preferably carried out by mixing.The incubation is preferably carried out for 5-15 minutes, morepreferably 10 minutes.

After incubation with LB, the liquid phase my be separated and the solidphase discarded. An activated charcoal may be added to the resultingliquid phase. Preferably, the activated charcoal is at a concentrationof 10-20 mg/ml, more preferably 30 mg/ml. The solution may then be mixedfor 5-15 minutes, preferably 10 minutes. The charcoal and otherparticulate are then removed e.g. by filtration.

An alcohol solution is added to, mixed with, or incubated with thesolution in embodiments of the invention. An alcohol solution comprisesat least one alcohol. The alcohol solution can be about, be at leastabout, or be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100% alcohol, or any range therein.In certain embodiments, it is added to a powder to make the finalsolution have a concentration of alcohol of about, about at least, orabout at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90%, orany range therein. In specific embodiments, the amount of alcoholsolution added to the powder gives it an alcohol concentration of 60%.Alcohols include, but are not limited to, ethanol, propanol,isopropanol, and methanol, with isopropanol as preferred. An alcoholsolution may be also used in additional steps of methods of theinvention to precipitate RNA.

Isopropanol is preferably added at a concentration of 100%.

Purification of small RNAs from the solution includes using a solidsupport, such as a mineral or polymer support.

Essential according to the invention is the use of a nucleic acid (NA)binding solid support e.g. silica particles capable of binding the NA inthe presence of a chaotropic substance. By silica are meant SiO2crystals and other forms of silicon oxide, such skeletons of diatomsbuilt up from SiO2, amorphous silicon oxide and glass powder. Alsoalkylsilica, aluminum silicate (zeolite), activated silica with —NH2,latex particles, certain polymeric materials forming the inside wall ofa cuvette or a microtiter plate, or filter materials for exampleconsisting of nitrocellulose are suitable as nucleic acid binding solidphase according to the invention.

A “solid support” or “support” refers to a physical structure containinga material which contacts the solution and that does not irreversiblyreact to macromolecules in the solution, particularly with small RNAmolecules. In particular embodiments, the solid support binds small RNAmolecules; in additional cases, it binds small RNA molecules, but doesnot bind one or more other types of macromolecules in the sample. Thematerial in the solid support may include a mineral or polymer, in whichcase the support is referred to as a “mineral or polymer support.”

Mineral or polymer supports include supports involving silica. Supportsinclude, but are not limited to, beads, columns and filters. In furtherembodiments, the mineral or polymer support is a silica filter orcolumn.

In a preferred embodiment, magnetic silica beads are added to thesolution, preferably at a concentration of 1-5 mg/mL, more preferably ata concentration of 2.5 mg/mL.

In a more preferred embodiment, the magnetic silica beads are basicmagprep silica [Merck Millipore], having a diameter of about 1 μm, acomposition of >95% magnetite, a surface area of 16-22 m2/mg, a bindingcapacity of >10 μg DNA or RNA per mg.

The incubation with silica beads preferably lasts 10 minutes and iscarried out by gentle shaking.

The magnetic beads are then preferably captured with magnete and theliquid is diascarded.

In an alternative embodiment, the solution is poured through a filter,preferably a muslin filter, into a Perspex column comprising silica gel,preferably at a concentration of 1.5 g/gDW tissue.

In particular, amorphous silica Sigma 274739 (50-70 mesh) wasresuspended with 10% HCl, and allowed to settle for 24 h. Thesupernatant was aspirated and discarded. The pellet was resuspended with6 ml of 0.1 M HCl and then aliquoted and stored at 4 C.

In a preferred embodiment, after addition of isopropanol, the liquidphase is pured into a column containing 1-5 μm diameter silicon dioxidebeads, preferably 5 mg/g tissue. In an alternative embodiment, 1-5 μmdiameter silicon dioxide beads, preferably 100 mg/g tissue are addedafter the addition of isopropanol and preferably agitated for 5-15minutes, preferably for 10 minutes. The liquid phase may then beremoved.

Alternatively, in some embodiments, the mineral or polymer support mayinclude polymers or nonpolymers with electronegative groups. In someembodiments, the material is or has polyacrylate, polystyrene, latex,polyacrylonitrile, polyvinylchloride, methacrylate, and/or methylmethacrylate.

After a solution is applied or mixed with a solid support, the materialmay be washed with a solution. In some embodiments, a mineral or polymersupport is washed with a first wash solution comprising alcohol, afterapplying the lysate to the mineral or polymer support. The methods ofthe invention may involve 1, 2, 3, 4, 5 or more washes with the washsolution. The wash solution used when more than one washing is involvedmay be the same or different. It is generally understood that moleculesthat come through the material in a wash cycle are discarded.

In a preferred embodiment, beads or columns are washed three times witha 80% or 85% solution of ethanol.

In an alternative embodiment, the column is washed with 10 volumes of a80% solution of ethanol.

Beads or column are then preferably dried to remove residual ethanol.

In other methods of the invention, the desired RNA molecules are elutedfrom the solid support.

In certain embodiments, small RNA molecules are eluted from a solidsupport such as a mineral or polymer support at a temperature of about60° C. to about 100° C. It is contemplated that the temperature at whichthe RNA molecules are eluted is about or at least about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100° C. ormore, or any range therein. The molecules may be eluted with any elutionsolution. In some embodiments, the elution solution is an ionicsolution, that is, it includes ions. In particular embodiments, theelution solution includes up to 10 mM salt. It is contemplated to beabout, at least about, or at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more mM salt. In certain embodiments, the salt consists of acombination of Li+, Na+, K+, or NH4+ as cation and Cl—, Br, I—,ethylenediaminetetraacetate, or citrate as anion.

In a preferred embodiment of the method of the invention, the sRNA areeluted with RNAse free water, more preferably at a temperature of 65° C.Volume range for the water for the beads is 60-200 uL/mg beads (0.6-2:1v/W). For the silica gel the volume range is 1-5 mL/g resin (1-5:1 v/W).In a preferred embodiment, water is used at a concentration of 1 Volumeor of 125 μL/mg of beads.

The present method may be used also for isolating small RNA from plants.

The present invention also concerns kits for isolating small RNAmolecule (or fractions enriched of small RNA molecules), such as miRNAand/or siRNA from a fungi or plant sample, particularly a cell or tissuesample. Thus, any of the compositions discussed above can be includedwith any other composition discussed above for inclusion in a kit. Insome embodiments, there are kits for isolating small RNA moleculescomprising: a) isopropanol, b) one or more small RNA wash solution (s),and e) an elution solution.

In preferred embodiment, the kit contains: a) lysis buffer; b)isopropanol ; c) a wash solution comprising 80% ethanol; f) an elutionsolution comprising RNA-se free water; g) a gel loading buffer II; h)collection tubes; and i) filter cartridges.

In some embodiments, kits of the invention include one or more of thefollowing in a suitable container means (consistent with compositionsdiscussed above): a silica filter or column; elution buffer; washbuffer; alcohol solution; and RNase inhibitor.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit (they maybe packaged together), the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits of the present invention also willtypically include a means for containing the RNA, and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained. When the components of the kit are providedin one and/or more liquid solutions, the liquid solution is an aqueoussolution, with a sterile aqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder (s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans. The container means will generally include at least one vial,test tube, flask, bottle, syringe and/or other container means, intowhich the nucleic acid formulations are placed, preferably, suitablyallocated. The kits may also comprise a second container means forcontaining a sterile, pharmaceutically acceptable buffer and/or otherdiluent.

Such kits may also include components that preserve or maintain the RNAor that protect against its degradation. Such components may beRNAse-free or protect against RNAses. Such kits generally will comprise,in suitable means, distinct containers for each individual reagent orsolution.

The sRNA molecules obtained by the present method may be used in thefood or nutraceutical field. Food products or foodstuffs that mayinclude said sRNA molecules are for example beverages, for instancesport drinks, fruiting juices, and alcoholic drinks as well as liquidpreparation to be added to drinking water and liquid food. In anotherembodiment food products or foodstuffs comprise solid or semi-solidfoods, e.g. baked goods, puddings, dairy products, confections, snackfoods, or frozen confections or novelties (e.g., ice cream, milkshakes), prepared frozen meals, candy, snack products (e.g., chips),liquid food such as soups, spreads, sauces, salad dressings, preparedmeat products, cheese, yogurt and any other fat or oil containing foods,and food ingredients (e.g., wheat flour). The term food products orfoodstuffs also includes functional foods and prepared food products,the latter referring to any pre-packaged food approved for humanconsumption.

The resulting food products or foodstuffs contain an enlarged amountand/or concentration of plant or fungal sRNA molecules compared to thefood products or foodstuffs without addition of such food additive.

In a preferred embodiment, such food products or foodstuffs contain anamount of plant or fungal sRNA or sRNA molecules which is at least 10%higher than the amount in the food product or foodstuff without additionof such food additive comprising a sRNA molecules, more preferably theamount is at least 20%, 50%, 100% or 200% higher than in the foodproduct or foodstuff without addition of such food additive.

Food compositions and dietary supplements are preferably administeredorally. Dietary supplements may be delivered in any suitable format,preferably for oral delivery. The ingredients of the dietary supplementof this invention are acceptable excipients and/or carriers for oralconsumption. The carrier may be a liquid, gel, gelcap, capsule, powder,solid tablet (coated or non-coated), tea, or the like.

In other embodiments, the dietary supplement is provided as a powder orliquid suitable for adding by the consumer to a food or beverage. Forexample, in some embodiments, the dietary supplement can be administeredto an individual in the form of a powder, for instance to be used bymixing into a beverage, or by stirring into a semi-solid food, or byotherwise adding to a food e.g. enclosed in caps of food or beveragecontainer for release immediately before consumption.

The dosage of composition comprising sRNA molecules of the invention asfood additive vary depending upon known factors, such as its mode androute of administration; the age, health and weight of the recipient;the nature and extent of the symptoms; the kind of concurrent treatment;

the frequency of treatment; and the effect desired which can bedetermined by the expert in the field with normal trials. Typically,dosage amounts of the plant or fungal sRNA molecules may vary from 10-20mg sRNA molecules per application to 1-2 mg sRNA molecules for dailyuse/application.

The dietary supplement may be administered as single dose or multipledoses.

Suitable routes of administration of the pharmaceutical composition ofthe invention include, for example, oral, intranasal and parenteraladministration.

The pharmaceutical composition of the present invention can beadministered in the form of a dosage unit, for example tablets orcapsules, or a solution.

Suitable pharmaceutical carriers are e.g. described in Remington'sPharmaceutical Sciences, a standard reference text in this field. Thepharmaceutical composition may further comprise conventionalpharmaceutical additives and adjuvants, excipients or diluents,including, but not limited to, water, gelatin of any origin, vegetablegums, ligninsulfonate, talc, sugars, starch, gum arabic, vegetable oils,polyalkylene glycols, flavoring agents, preservatives, stabilizers,emulsifying agents, buffers, lubricants, colorants, wetting agents,fillers, and the like.

The pharmaceutical compositions may be administered as single dose ormultiple doses.

The compositions according to the present invention may be in anygalenic form that is suitable for administering to the animal bodyincluding the human body, more in particular in any form that isconventional for oral administration, e.g. in solid form, for exampletablets, pills, granules, dragees, capsules, and effervescentformulations such as powders and tablets, or in liquid form, forinstance in the form of solutions, emulsions or suspensions, for exampleas pastes and oily suspensions. The pastes may be filled into hard orsoft shell capsules, whereby the capsules feature e.g. a matrix of(fish, swine, poultry, cow) gelatin, plant proteins or lignin sulfonate.Examples for other application forms are forms for transdermal,parenteral, topical or injectable administration. The pharmaceuticalcompositions may be in the form of controlled (delayed) releaseformulations. Topical formulation may contain e.g. ointments, creams,gels, lotions, solutions. In the present invention the term “effectiveamount” shall mean an amount which achieves a desired effect ortherapeutic effect as such effect is understood by those of ordinaryskill in the art. For injection, including, without limitation,intravenous, intramuscular and subcutaneous injection, the compounds ofthe invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as physiological saline bufferor polar solvents including, without limitation, a pyrrolidone ordimethylsulfoxide.

Formulations for injection may be presented in unit dosage form, e.g. inampoules or in multi-dose containers. Pharmaceutical compositions forparenteral administration include aqueous solutions of a water solubleform, such as, without limitation, a salt of the active compound.Additionally, suspensions of the active compounds may be prepared in alipophilic vehicle. Suitable lipophilic vehicles include fatty oils suchas sesame oil, synthetic fatty acid esters such as ethyl oleate andtriglycerides, or materials such as liposomes. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxym ethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers and/or agents that increase the solubility of the compoundsto allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use. For oral administration, the compounds can be formulated bycombining the active compounds with pharmaceutically acceptable carrierswell-known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, lozenges, dragees,capsules, liquids, gels, syrups, pastes, slurries, solutions,suspensions, concentrated solutions and suspensions for diluting in thedrinking water of a patient, premixes for dilution in the feed of apatient, and the like, for oral ingestion by a patient. Pharmaceuticalpreparations for oral use can be made using a solid excipient,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding other suitable auxiliaries if desired, to obtaintablets or dragee cores. Useful excipients are, in particular, fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol,cellulose preparations such as, for example, maize starch, wheat starch,rice starch and potato starch and other materials such as gelatin, gumtragacanth, methyl cellulose, hydroxypropyl-methylcellulose, sodiumcarboxy- methylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid. A salt such as sodium alginate mayalso be used.

For administration by inhalation, the sRNA molecules of the presentinvention can conveniently be delivered in the form of an aerosol sprayusing a pressurized pack or a nebulizer and a suitable propellant. ThesRNA molecules may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the sRNA moleculesmay also be formulated as depot preparations. Such long actingformulations may be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thecompounds of this invention may be formulated for this route ofadministration with suitable polymeric or hydrophobic materials (forinstance, in an emulsion with a pharmacologically acceptable oil), withion exchange resins, or as a sparingly soluble derivative such as,without limitation, a sparingly soluble salt.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedisclosure herein.

The administration of such sRNA molecules may occur through complexingof the sequence with a suitable delivery system such as complexationwith cationic liposomes or inclusion in nanovesicles.

The delivery system can then be formulated for oral (includingsub-lingual) administration as dietary supplement or directly integratedin food matrix or drinks for regular use. The daily doses may be in therange of 1-5 mg of active principle.

The delivery system may be formulated for topical administration. Thedaily doses may be in the range of 1-5 mg of active principle.

The delivery system may be formulated for intravenous administrationwith dosage over 10 mg of active principle.

The above defined sRNAs of 15-60 bp of length extracted from plants ormushrooms may be functionalized to enhance their bioavailability andanti-inflammatory efficacy When dietary vegetables are consumed throughthe diet a small fraction of the microRNAs originally present in thefood matrix passes through the CI tract and becomes available in thebloodstream (0.05%-0.5%).

Such reduced bioavailability limits the anti-inflammatory efficacytowards human cells and organs of miRNAs and sRNAs naturally absorbedthrough the diet.

Moreover, bioavailable dietary naked miRNAs or sRNAs may be subjected todegradation or structural modification within the bloodstream withensuing loss of bioactivity.

Inventors have discovered that complexation of 15-60 bp sRNAs extractedfrom plants or mushrooms with liposomes, exosomes or nanoparticlesenables their effective uptake by dendritic cells of the human immunesystem.

Moreover complexation of such bioactive compounds will preserveintegrity and reduce degradation in case of:

-   -   oral consumption: during the mastication and passage through the        GI tract.    -   topic application: during application on skin surface and        permeation in the dermis    -   intravenous application: for movement within the bloodstream and        transfer to recipient cells and organs

In case of oral consumption or topic application, complexation will alsofacilitate their transfer into the bloodstream.

Complexation with cationic liposomes occurs naturally byphysico-chemical interaction sRNAs are negatively charged in solution.

In a preferred embodiment the sRNAs as described above are providedwithin a delivery vehicle, optionally wherein the delivery vehicle isselected from liposomes, particularly cationic liposomes, liposomecomprising lipids, or nanovesicles. Many methods are available forpreparing liposomes, as described in, for example, Szoka et al., Ann.Rev. Biophys. Bioeng., 9:467 (1980) and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369.

In an embodiment of the invention, at least part of the lipids of theliposomes are selected from the group consisting of phospholipids,sterols and sterol derivatives.

In a particular embodiment of the invention wherein the lipid of theliposomes comprises or constitutes a member selected from the groupconsisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol(PI), phosphatidic acid (PA), DPG (bisphosphatidyl glycerol), PEOH(phosphatidyl alcohol), cholesterol, ergosterol and lanosterol. In afurther embodiment, the liposome comprises phosphatidylcholines selectedfrom the group consisting of 1,2-dioleoyl-phosphatidylcholine,1,2-dipalmitoyl-phosphatidylcholine,1,2-dimyristoyl-phosphatidylcholine, 1,2-distearoyl-phosphatidylcholine,1-oleoyl-2-palmitoyl-phosphatidylcholine, 1-oleoyl-2-stearoyl-phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholineand 1-stearoyl-2-oleoyl-phosphatidylcholine.

In an embodiment of the inventions the liposome comprisesphosphatidylethanolamines selected from the group consisting of1,2-dioleoyl-phosphatidylethanolamine,1,2-dipalmitoyl-phosphatidylethanolamine,1,2-dimyristoyl-phosphatidylethanolamine,1,2-distearoyl-phosphatidylethanolamine,1-oleoyl-2-palmitoyl-phosphatidylethanolamine, 1-oleoyl-2-stearoyl-phosphatidylethanolamine,1-palmitoyl-2-oleoyl-phosphatidylethanolamine,1-stearoyl-2-oleoyl-phosphatidylethanolamine andN-succinyl-dioleoyl-phosphatidylethanolamine; the phosphatidylserinesare selected from the group consisting 1,2-dioleoyl-phosphatidylserine,1,2-dipalmitoyl-phosphatidylserine, 1,2-dimyristoyl-phosphatidylserine,1,2-distearoyl-phosphatidylserine,1-oleoyl-2-palmitoyl-phosphatidylserine,1-oleoyl-2-stearoyl-phosphatidylserine,1-palmitoyl-2-oleoyl-phosphatidylserine and1-stearoyl-2-oleoyl-phosphatidylserine; the phosphatidylglycerols areselected from the group consisting 1,2-dioleoyl-phosphatidylglycerol,1,2-dipalmitoyl-phosphatidylglycerol,1,2-dimyristoyl-phosphatidylglycerol,1,2-distearoyl-phosphatidylglycerol,1-oleoyl-2-palmitoyl-phosphatidylglycerol,1-oleoyl-2-stearoyl-phosphatidylglycerol,1-palmitoyl-2-oleoyl-phosphatidylglycerol and1-stearoyl-2-oleoyl-phosphatidylglycerol; the phosphatidic acids areselected from the group consisting of di-palmitoyl-glycerophosphatidicacid, di-stearoyl-glycerophosphatidic acid,di-myrostoyl-glycerophosphatidic acid, di-oleoyl-glycerophosphatidicacid, palmitoyl-oleoyl-glycerophosphatidic acid.

In nnother embodiment of the liposome of the invention, the lipidcomprises or constitutes phosphatidylglycerol (PG),phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid(PA), DPG (bisphosphatidyl glycerol), PEOH (phosphatidyl alcohol),cholesterol, phosphatidylcholines such as1,2-dioleoyl-phosphatidylcholine, 1,2-dipalmitoyl-phosphatidylcholine,1,2-dimyristoyl-phosphatidylcholine, 1,2-distearoyl-phosphatidylcholine,1-oleoyl-2-palmitoyl-phosphatidylcholine,1-oleoyl-2-stearoyl-phosphatidylcholine,1-palmitoyl-2-oleoyl-phosphatidylcholine and1-stearoyl-2-oleoyl-phosphatidylcholine; phosphatidylethanolamines suchas 1,2-dioleoyl-phosphatidylethanolamine,1,2-dipalmitoyl-phosphatidylethanolamine,1,2-dimyristoyl-phosphatidylethanolamine,1,2-distearoyl-phosphatidylethanolamine,1-oleoyl-2-palmitoyl-phosphatidylethanolamine,1-oleoyl-2-stearoyl-phosphatidylethanolamine,1-palmitoyl-2-oleoyl-phosphatidylethanolamine,1-stearoyl-2-oleoyl-phosphatidylethanolamine andN-succinyl-dioleoyl-phosphatidylethanolamine; phosphatidylserines suchas 1,2-dioleoyl-phosphatidylserine, 1,2-dipalmitoyl-phosphatidylserine,1,2-dimyristoyl-phosphatidylserine, 1,2-distearoyl-phosphatidylserine,1-oleoyl-2-palmitoyl-phosphatidylserine,1-oleoyl-2-stearoyl-phosphatidylserine,1-palmitoyl-2-oleoyl-phosphatidylserine and1-stearoyl-2-oleoyl-phosphatidylserine; phosphatidylglycerols such as1,2-dioleoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol,1,2-dimyristoyl-phosphatidylglycerol,1,2-distearoyl-phosphatidylglycerol,1-oleoyl-2-palmitoyl-phosphatidylglycerol,1-oleoyl-2-stearoyl-phosphatidylglycerol,1-palmitoyl-2-oleoyl-phosphatidylglycerol and1-stearoyl-2-oleoyl-phosphatidylglycerol;1,2-dioctadecanoyl-sn-glycero-3-ethylphosphocholine (Ethyl PC);pegylated lipids; pegylated phospoholipids such asphophatidylethanolamine-N-[methoxy(polyethyleneglycol)-1000],phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000],phophatidylethanolamine-N-[methoxy(polyethylene glycol)-3000],phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-5000]; pegylatedceramides such asN-octanoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)1000]},N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]},N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)3000]},N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)5000]};lyso-phosphatidylcholines, lyso-phosphatidylethanolamines,lyso-phosphatidylglycerols, lyso-phosphatidylserines, ceramides;

sphingolipids; glycolipids such as ganglioside GMI; glucolipids;sulphatides; phosphatidic acid, such as di-palmitoyl-glycerophosphatidicacid; palmitic fatty acids; stearic fatty acids; arachidonic fattyacids; lauric fatty acids; myristic fatty acids; lauroleic fatty acids;physeteric fatty acids; myristoleic fatty acids; palmitoleic fattyacids; petroselinic fatty acids; oleic fatty acids; isolauric fattyacids; isomyristic fatty acids; isostearic fatty acids; sterol andsterol derivatives such as cholesterol, cholesterol hemisuccinate,cholesterol sulphate, and cholesteryl-(4-trimethylammonio)-butanoate,ergosterol, lanosterol; polyoxyethylene fatty acids esters andpolyoxyethylene fatty acids alcohols; polyoxyethylene fatty acidsalcohol ethers; polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxy-stearate; glycerol polyethylene glycolricinoleate; ethoxylated soybean sterols; ethoxylated castor oil;polyoxyethylene polyoxypropylene fatty acid polymers; polyoxyethylenefatty acid stearates; di-oleoyl-sn-glycerol;dipalmitoyl-succinylglycerol; 1,3-dipalmitoyl-2-succinylglycerol;1-alkyl-2-acyl-phosphatidylcholines such as1-hexadecyl-2-palmitoyl-phosphatidylcholine;1-alkyl-2-acyl-phosphatidylethanolamines such as1-hexadecyl-2-palmitoyl-phosphatidylethanolamine;1-alkyl-2-acyl-phosphatidylserines such as1-hexadecyl-2-palmitoyl-phosphatidylserine;1-alkyl-2-acyl-phosphatidylglycerols such as1-hexadecyl-2-palmitoyl-phosphatidylglycerol;1-alkyl-2-alkyl-phosphatidylcholines such as1-hexadecyl-2-hexadecyl-phosphatidylcholine;1-alkyl-2-alkyl-phosphatidylethanolamines such as1-hexadecyl-2-hexadecyl-phosphatidylethanolamine;1-alkyl-2-alkyl-phosphatidylserines such as1-hexadecyl-2-hexadecyl-phosphatidylserine;1-alkyl-2-alkyl-phosphatidylglycerols such as1-hexadecyl-2-hexadecyl-phosphatidylglycerol; andN-Succinyl-dioctadecylamine; palmitoylhomocysteine.

An embodiment of the invention is a liposome, wherein at least part ofthe lipids is a cationic lipid.

An embodiment of the invention is a liposome, wherein the cationiclipids are selected from the group consisting of stearylamine (SA),lauryltrimethylammonium bromide; cetyltrimethyl-ammonium bromide,myristyl trimethylammonium bromide, dimethyldioctadecylammonium bromide(DDAB), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] chole sterol(DC-Cholesterol), 1,2-ditetradecanoyl-3-trimethylammonium-propane(DMTAP), 1,2-dioctadecanoyl-3-trimethylammonium-propane (DOTAP) andDOTAP derivatives such as 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and1,2-dihexadecanoyl-3-trimethylammonium-propane,1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane (DODAP) and DODAPderivatives such as 1,2-ditetradecanoyl-3-dimethylammonium-propane,1,2-dihexadecanoyl-3-dimethylammonium-propane, and1,2-dioctadecanoyl-3-dimethylammonium-propane,1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),1,2-dioleoyl-c-(4′-trimethylammonium)-butanoyl-sn-glycerol (DOTB),dioctadecylamide-glycylspermine, SAINT-2, polycationic lipid2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), and GL67TM.

A particular embodiment of the invention is a liposome, wherein thecationic lipids are selected from the group consisting of stearylamine(SA), 1,2-dioctadecanoyl-3-trimethylammonium-propane (DOTAP) and1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane (DODAP), preferably1,2-dioctadecanoyl-3-trimethylammonium-propane (DOTAP).

Preferred cationic lipids are DOTAP and DOTAP derivatives. Additionalexamples of cationic lipids and lipid components may be found in or madeaccording to U.S. Pat. No. 4,804,539.

An embodiment of the invention is a liposome, wherein at least part ofthe lipids is a cationic lipopeptide selected from the group consistingof a lipid polyarginine conjugate, a lipid TAT conjugate, a lipidpolylysine conjugate, or a cationic liposaccharide or lipopolysaccharidesuch as a lipid chitosan conjugate.

In a preferred embodiment, the lipids are capable of forming a liposome.In particular, cationic lipids are suitable for this purpose.

Cationic lipids preferably include DOTAP, DOPE, DC-Chol/DOPE, DOTMA, andDOTMA/DOPE.

Cationic Lipids: Cationic lipids carry a net positive charge at aboutphysiological pH. Suitable cationic lipids include, but are not limitedto, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxyl)propyl-N,N-N-triethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”) ;1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxyl)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL). In particular embodiments, acationic lipid is an amino lipid.

Additional cationic lipids include1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA)1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1-Linoleoyl-2-linoeyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

An embodiment of the invention is a liposome or a vescicle, with aparticle size of between 0.1 and 100 um

In certain embodiments, the ratio of total lipid to sRNA is from 5 to 35(i.e. from 5 to 1 to 35 to 1, lipid weight to sRNA weight). In certainembodiments, the ratio of total lipid to sRNA is from 5 to 15 (i.e. from5 to 1 to 15 to 1, lipid weight to sRNA weight).

In a yet further preferred embodiment, exosomes may be used. Suchexosomes and their preparation are described e.g. in Montecalvo et al.(2012, Blood, 119: 756-766, and Stoorvogel, 2012, Blood, 119: 646-648).For example, exosomes loaded with plant or fungal sRNA or sRNAextract(s) or compositions comprising 2 or more plant or fungal sRNA orsRNA extract(s) may be used. For example, exosomes loaded with plantmiRNA may be used.

Polymeric nanoparticles formed by self-assembly of polycations withsiRNA can be used for extracellular delivery, cellular uptake andintracellular trafficking as a strategy to improve the therapeuticpotential of siRNA. Polycationic polymer-based nanoparticle (orpolyplex) systems used for site-specific delivery, cellular uptake andintracellular trafficking of siRNA. Such nanoparticles and in particularchitosan-based particles and their preparations are described e.g. inØstergaard et al., Therapeutic Applications of RNAi: Methods andProtocols, Humana Press 2009.

For example, nanoparticles loaded with plant or fungal sRNA or sRNAextract(s) or compositions comprising 2 or more plant or fungal sRNA orsRNA extract(s) may be used.

Another delivery strategy is via β1,3-D-glucan particles (GP), hollowand porous microspheres derived from Saccharomyces cerevisiae (Baker'syeast) that provide an efficient system for encapsulation, protection,and oral or systemic macrophage-targeted delivery of macromolecules,such as DNA, siRNA and proteins using either a polyplex orlayer-by-layer (LbL) synthesis methods. Such particles and theirpreparations are described e.g. in Soto and Ostroff, Nanomaterials forBiomedicine, 3, 57-79 2012. In a particular embodiement, plant or fungalsRNA is encapsulated in 2-4 μm hollow β1,3-D-glucan particles.

For example glucan particles loaded with plant or fungal sRNA moleculesof the invention may be used.

The “MRG extraction method” allows selective extraction and purificationof low molecular weight ribonucleic acid species (sRNAs) from plantsand/or mushroom-derived biomass which display anti-inflammatoryproperties (Plant microRNAs as novel immunomodulatory reagents.Scientific Reports 2016 Cavalieri et al.).Unlike other existing methods,which have all been designed for analytical purposes, the MRG protocolaims at the production of sRNA extracts for the food industry and doesnot involve the use of solvents or toxic reagents and achieve theselective extraction of sRNA from extracts through ionic interactionwith activated silica gel. It is important to note that the MRG methodaims at the selective enrichment in the extracts of the microRNA andsiRNA fraction which comprise small RNA of length between 19 and 24 nt.

These species are present both in the single stranded and doublestranded configurations (ssRNAs and dsRNAs) and are characterized by thepresence of a phosphate group at the 5′ ends and a methyl group at the3′ ends. These properties make these molecular species (particularly thedouble stranded configurations) much more resistant than other sRNAs todegradation during extraction.

The present invention will be illustrated by means of non limitingexamples in reference to the following figures.

FIG. 1. A method for lab-scale purification of sRNA from fungi.

FIG. 2. Electropherograms of sRNA extracts obtained using LB withdifferent pH.

FIG. 3. Electropherograms of sRNA extracts obtained using different SDSconcentrations in the LB.

FIG. 4. Electropherograms of sRNA extracts obtained using different NaClconcentrations in the LB

FIG. 5. Electropherograms of sRNA extracts obtained using different EDTAconcentrations in the LB.

FIG. 6. Total sRNA extracted from various plants and fungal tissues (100mg) using the “MRG sRNA extraction method”. The total sRNA content ofthe DW eluate from the silicon dioxide beads was determined using theAgilent RNA 6000 Nano Kit through the Bioanalyzer instrument /AgilentTechnologies) and data are expressed as ug/g of tissue ±S.D.

FIG. 7. MRG sRNA extraction method (column) from plant or fungi

FIG. 8. MRG sRNA extraction method (no column) from plant or fungi

FIG. 9. Exctraction in diluted bicarbonate solution

FIG. 10. The figure shows the RNA profile of concentrated sRNA extractsbefore (A) and after (B) RNAse A treatment using the Agilent RNA 6000Nano Kit through the Bioanalyzer instrument (Agilent Technologies).

FIG. 11. The figure shows the RNA profile of concentrated sRNA extractsbefore (A) and after (B) p19siRNA binding protein treatment using theAgilent RNA 6000 Nano Kit through the Bioanalyzer instrument (AgilentTechnologies)

EXAMPLES Materials and Method

Starting protocol for the extraction of sRNA from mushroom tissue basedon the utilization of non-toxic reagents for tissue lysis and magneticsilica beads for the isolation of sRNA from the extraction solution.

Basic Extraction Protocol (BEP):

1. Extraction of sRNA: 100 mg of mushroom tissue (fruit body of Agaricusbisporus) is incubated with 200 μL of pre-heated (55° C.) lysis buffer(LB) at room T for 10 min. with vortexing (30 sec. every 2 min).

2. Binding of sRNA to magnetic beads: after centrifugation, the extract(200 μL) is transferred to mμLtiwell dishes and isopropanol is added.Subsequently, 0.5 mg of magnetic silica beads (MagPrep basic, MerckMillipore) is added to the wells which are left gently shaking for 10min.

3. Washing of the beads: the beads are removed with a magnetic supportand the remaining solution is discarded. The beads are then washed threetimes with 85% ethanol and then left drying to remove residual ethanol.

4. Elution of sRNA: bound sRNA is removed from the beads with 60 μL ofpre-heated RNAse-free water.

Protocol Optimisation

Step 1: Varying LB Buffer pH

Conditions used: BEP with 60% isopropanol final concentration and LBwith different pH (pH 5-10) (see FIG. 2).

TABLE 1 Amount of sRNA extracted from mushroom using LB with pH: sRNAextracted pH of Lysis Buffer (ug/gDW) 5 n.d. 6 2 7 5 7.5 50 8 80 8.5 1009 5 9.5 n.d. 10 n.d.

The highest yield of sRNA was obtained using a LB with pH 8.5

Step 2: Varying SDS Concentrations in the LB.

Conditions used: BEP with 60% isopropanol final concentration and LB atpH 8.5 and various SDS concentration (0-6%) (see FIG. 3).

TABLE 2 Amount of sRNA extracted from mushroom tissue using various SDSconcentrations in the LB: SDS concentration sRNA extracted in LB(ug/gDW) 0% 20 2% 80 4% 65 6% 30

The highest yield of sRNA was obtained with 2% SDS concentration in theLB

Step 3: Varying NaCl Concentration in the LB.

Conditions used: BEP with 60% isopropanol final con centration and LB atpH8.5, 2% SDS and different NaCl concentrations (0-0.3M) (see FIG. 4)

TABLE 3 Amount of sRNA extracted from mushroom tissue using various NaCLconcentrations: NaCl concentration sRNA extracted in LB (ug/gDW)  0M 200.1M 70 0.2M 100 0.3M 90

The highest yield of sRNA was obtained with 0.2M NaCl in the LB

Step 4: Varying EDTA Concentration in the LB.

Conditions used: BEP with 60% isopropanol final con centration and LB atpH8.5, 2% SDS, 0.2 M NaCl and different EDTA concentrations (0.20 mM)(see FIG. 5).

TABLE 4 Amount of sRNA extracted from mushroom tissue using various EDTAconcentrations in the LB: EDTA concentration sRNA extracted in LB(ug/gDW) 0M n.d. 10 mM 100 20 mM 100

The highest yield of sRNA was obtained with 10 or 20 mM EDTAconcentration in the LB.

Optimized extraction protocol (OEP):

1. Extraction of sRNA: 100 mg of mushroom tissue (fruit body of Agaricusbisporus) is incubated with 200 μL of pre-heated (55° C.) lysis buffer(LB: 50 mM Tris-HCl (pH 8.5) containing 0.2M NaCl, 10 mM EDTA and 2%SDS) at room T for 10 min. with vortexing (30 sec. every 2 min).

2. Binding of sRNA to magnetic beads: after centrifugation, the extract(200 μL) is transferred to mμLtiwell dishes and 300 μL isopropanol(100%) is added. Subsequently, 0.5 mg of magnetic silica beads (MagPrepbasic, Merck Millipore) is added to the wells which are left gentlyshaking for 10 min.

3. Washing of the beads: the beads are removed with a magnetic supportand the remaining solution is discarded. The beads are then washed threetimes with 85% ethanol and then left drying to remove residual ethanol.

4. Elution of sRNA: bound sRNA is removed from the beads with 60 μL ofpre-heated RNAse-free water.

With this OEP, a yield of 100 μg/g FW small RNA from Agaricus bisporusis obtained.

MRG Extraction Method

Extraction Buffer:

Tris-HCl 50 mM pH 8.5 NaCl 0.2M EDTA 10 mM pH 8.5 SDS 2% v/v

-   -   1) Place 100 mg of tissue finely grounded tissue (liquid        nitrogen) in a 2 ml vessel    -   2) Add extraction pre-heated (55° C.) buffer (200 μl) and mix        vigoroulsy (vortex)    -   3) Incubate (10′, RT) in thermomixer with a shaking regime of        10″ every 2′ at 400 RPM    -   4) Spin ((10′ @ 09300 RCF, RT)    -   5) Transfer the supernatant in una new 2 ml vessel    -   6) Add isopropanol (300 μl) and mix    -   7) Add 15 mg di silica gel (Sigma S-5631, prewashed×2 in DW and        incubated O/N with 0.1M HCl) and mix    -   8) Incubate 10′ at RT with gentle shaking    -   9) Spin (10′ @ 9300 RCF, RT)    -   10) Discard the supernatant and add 500 μl 85% EtOH. Mix gently    -   11) Spin (5′ @ 9300 RCF, RT)    -   12) Repeat×2 step 10 -11    -   13) Discard the supernatant and allow the silica gel to dry up.    -   14) Add 60 μl DW    -   15) Spin (5′ @ 9300 RCF, RT) and collect the supernatant

Comparison between known extraction methods and the method of theinvention

Here inventors compare extraction efficiency and purity of the finalextract between the MRG extraction method and other protocols whichcontain compounds that can aid the purifications of the sRNA fractionsuch as NaBO4 (Borax) or CTAB which bind to polymeric molecules such aspolysaccharides, proteins and DNA or LiCl which at high concentrations(≥2M) precipitate high molecular weight nucleic acids (RNA and DNA)species above 150 bps. The method of the invention is a scalable,inexpensive and safe method for the industrial production of sRNAsextracts for the food industry. All existing protocols have beenmodified to remove highly toxic compounds and minimize the numberpurification steps.

Here is a list of the methods used:

Modified CTAB method (Gambino, G., Perrone, I. and Gribaudo, I. (2008),A Rapid and effective method for RNA extraction from different tissuesof grapevine and other woody plants.

Phytochem. Anal., 19: 520-525)

Extraction Buffer Composition:

CTAB 2% (w/v) EDTA 20 mM pH 8.5 NaCl 1.4M Tris-HCl 100 mM pH 8.5 PVP 25%(w/v)

-   -   1) Place 150 mg of tissue finely grounded tissue (liquid        nitrogen) in a 1.5 ml vessel    -   2) Add pre-heated (65° C.) extraction buffer (600 μl) and mix        vigorously (vortex)    -   3) Incubate (65° C., 10′) in thermomixer with a shaking regime        of 20″ every 2′ at 400 RPM    -   4) Spin (10′ @ 9300 RCF, 18° C.)    -   5) Transfer supernatant to a new 1.5 ml vessel    -   6) Add a volume of 5M NaCl solution to reach 0.5M final NaC1        concentration and mix    -   7) Add a volume of 8M LiCl solution to reach a 2M final LiC1        concentration and mix    -   8) Incubate (4° C., 3.5 h)    -   9) Spin (15′ @ 16500 RCF, 4° C.)    -   10) Transfer the supernatant to a new 2 ml vessel    -   11) Add a volume of 3M NaAc solution (pH 5.2) to reach a 0.3M        final NaAc concentration and mix.    -   12) Add an equal volume of isopropanol (50% v/v) and mix    -   13) Incubate in ice-cold water for 1 h    -   14) Spin (15′ @ 16500 RCF, 4° C.)    -   15) Discard the supernatant and resuspend the pellet in 500 μl        80% EtOH    -   16) Spin (15′ @ 16500 RCF, 4° C.)    -   17) Discard the supernatant and allow the pellet to dry up    -   18) Add DW (50 μl)

LiCl method (modified from “Rosas-Cárdenas, F. de F., Escobar-Guzmán,R., Cruz-Hernández, A., Marsch-Martínez, N., & de Folter, S. (2015). Anefficient method for miRNA detection and localization in crop plants.Frontiers in Plant Science, 6, 99)

Extraction Buffer:

Tris-HCl 100 mM, pH 8.5 SDS 1% (w/v) LiCl 100, mM EDTA 10 mM, pH 8.5

-   -   1) Place 100 mg of tissue finely grounded tissue (liquid        nitrogen) in a 2 ml vessel    -   2) Add extraction buffer (500 μl) and mix vigorously (vortex)    -   3) Incubate (60° C., 10′) in thermomixer with a shaking regime        of 20″ every 2′ at 400 RPM    -   4) Spin (10′ @ 9300 RCF, 4° C.)    -   5) Transfer the supernatant to a new 2 ml vessel    -   6) Add 2M Add a volume of KCl 2M solution to reach 0.55 M final        KCl concentration    -   7) Add a volume of PEG 8000 (50% w/v) solution equal to 1/9 of        final volume and mix.    -   8) Incubate 10′ in ice-cold water    -   9) Add a volume of a 8M LiCl solution to reach 2M final LiCl        concentration    -   10) Incubate 30′ in ice-cold water    -   11) Spin (10′ @ 9300 RCF, 4° C.)    -   12) Transfer the supernatant to a new vessel    -   13) Add a volume of 3M NaAc solution (pH 5.2) to reach 0.5M        final NaAc concentration    -   14) Add 2 volumes of EtOH and mix    -   15) Incubate overnight at −20° C.    -   16) Spin ((15′ @ 16500 RCF, 4° C.)    -   17) Discard the supernatant and resuspend pellet in 600 μl 80%        EtOH    -   18) Spin (10′ @ 16500 RCF, 4°)    -   19) Discard the supernatant and allow the pellet to dry up    -   20) Add DW (50 μl)

XT Method (Moser, C., Gatto, P., Moser, M., Pindo and Velasco, R.Isolation of functional RNA from small amounts of different grape andapple tissues. Mol Biotechnol (2004) 26: 95). Extraction Buffer:

NaBO4 * 10 H2O 0.2M EDTA 30 mM SDS 1% v/v

Adjust to pH 9.0

Just before use add PVP40 (2% w/v final concentration) and Tween 20 (1%v/v final concentration)

-   -   1) Place 100 mg of tissue finely grounded tissue (liquid        nitrogen) in a 2 ml vessel    -   2) Add extraction pre-heated (65° C.) buffer (300 μl) and mix        vigoroulsy (vortex)    -   3) Incubate (65° C., 10′) in thermomixer with a shaking regime        of 20″ every 2′ at 400 RPM    -   4) Spin (10′ @ 9300 RCF, 4° C.)    -   5) Transfer the supernatant to a new 1.5 mL vessel    -   6) Add a volume of a 5M NaCl solution to reach 0.5M final NaCl        concentration and mix    -   7) Add a volume of 8M LiCl solution to reach 2M final LiCl        concentration    -   8) Incubate 3 h at 4° C.    -   9) Spin (15′ @ 16500 RCF, 4° C.)    -   10) Transfer the supernatant to a new 1.5 mL vessel)    -   11) Add a volume of 3M NaAc3M solution (pH 5.2) to reach 0.3M        final NaAc concentration and mix    -   12) Add 1 volume of isopropanol and mix vigorously    -   13) Incubate 1 h in ice-cold water    -   14) Spin (15′ @ 16500 RCF, 4° C.)    -   15) Discard the supernatant and wash the pellet with 500 μl 80%        EtOH    -   16) Spin (10′ @ 16500 RCF, 4° C.)    -   17) Discard the supernatant and allow the pellet to dry up    -   18) Add DW (50 μl)

TABLE 5 sRNA yield obtained with the above described methods: LiCl CTABXT MRG Bean mature 275 ± 38 41 ± 21 163 ± 54 533 ± 69 cotyledons Peacotyledons 164 ± 51 9 ± 7 371 ± 48 774 ± 56 Peppermint leaves  66 ± 1178 ± 14  98 ± 12 125 ± 75 (fresh) Peppermint leaves 133 ± 23 106 ± 15 125 ± 30  572 ± 129 (dry) Soybean sprouts 120 ± 20 87 ± 9  125 ± 20 314± 41 Carrot root 24 ± 4 5 ± 5 45 ± 5  19 ± 11 Calendula flowers  77 ± 11116 ± 77  80 ± 8 110 ± 10 Onion bulb 32 ± 4 6 ± 4  23 ± 13  59 ± 16Mushrooms  67 ± 10 55 ± 11 100 ± 10 146 ± 11 (Champignon) Cumin seed 12± 7 5 ± 3 24 ± 9 24 ± 8 Apple fruiting  5 ± 5 20 ± 6   8 ± 4 27 ± 9

Table 5. Total sRNA extracted from different tissues of plants and fungiusing different extraction methods. The sRNA content of the finalextract was determined using the Agilent RNA 6000 Nano Kit through theBioanalyzer instrument/Agilent Technologies) and data are expressed asug/g of tissue ±S.D.

MRG method scale up to 1 Kg range

The matrix chosen to setup the scale up of the MRG method was composedby the fruiting bodies (caps and stalks) of “champignon” mushrooms ormature cotyledons of beans or peas.

1 Kg of plant or mushroom tissue was snap frozen in liquid nitrogen andground to a fine powder using the TissueLyser II (Qiagen) with titaniumjars and 10 mm diameters titanium spheres. Jars and spheres were keptcold with liquid nitrogen. The grinding time was set to 1 minute at ashake frequency of 30 Hz.

The tissue was then placed in a 3 L becker and 2 L of pre-heated (55°C.) and MRG Lysis Buffer (LB) was slowly added and thoroughly mixed tohomogenize the material. The solution was kept under gentle agitationfor 10 minutes.

The homogenate was then filtered through cellulose sheets and theresulting liquid was transfered to a 5L tank to which 1.5 volumes of100% isopropanol were added. After agitation to homogenize the solution,100 gr of silicon dioxide beads, 50/70 mesh particle size (Sigma S-5631)were added and the tank was kept under agitation for 10 minutes. Thebeads were prewashed×2 in DW and incubated O/N with 0.1M HCl prior touse.

After allowing sedimentation of the beads, the liquid phase was removedand the beads were washed 3 times with 400 ml EtOH 85% which was addedto the beads and left in the tank with agitation for 10 minutes.

After the final wash the silicon dioxide was allowed to dry and thensRNAs were eluted with 200 mL of pre-heated (65° C.) water.

TABLE 6 Total sRNA extracted from 1 Kg of plants and fungal tissue usingthe MRG extraction method. The sRNA content of the final extract wasdetermined using the Agilent RNA 6000 Nano Kit through the Bioanalyzerinstrument/Agilent Technologies) and data are expressed as mg/Kg oftissue ± S.D. sRNA yield TISSUE (mg/Kg) Bean mature cotyledons 475 ± 78Pea mature cotyledons 564 ± 59 Mushrooms (champignon) 116 ± 31 fruitingbody

Extraction of sRNAs from plant or mushroom tissues without tissuehomogenisation.

A method for the extraction of sRNA from plant and mushroom tissueswithout tissue homogenization was also developed. Intact or coarselychopped plant or mushroom tissues (100 mg) were pre-incubated for 16 hat 4° C. in 8.75 mM NaHCO₃. Subsequently, after removal of the washingliquid, the tissues were placed in vessels containing 1L of variousextraction media: MRG LB (with exclusion of SDS), water or 30 mM NaHCO₃,and incubated at 60° C. or 95° C. (boiling) and the sRNA present in theliquid phase measured using the MRG method (from step 5). For incubationat 60° C. the liquid phase was exchanged every 60 min. and the totalduration was 180 min. For incubation at 95° C., the volume was keptconstant by topping up and the whole duration was 60 min.

TABLE 7 Extraction treatment MRG LB WATER NaHCO₃ TISSUE 95° C. 95° C.60° C. 95° C. Bean cotyledons 103 ± 19  87 ± 24 125 ± 21 447 ± 51 Peacotyledons 6 ± 2 24 ± 7   40 ± 15 363 ± 53 Peppermeant leaves 6 ± 2 15 ±2   7 ± 2 33 ± 5 (fresh) Peppermeant leaves 75 ± 21 54 ± 11 170 ± 22 170± 11 (dry) Soybean sprouts nd 2 ± 1 nd  3 ± 2 Carrot root nd 4 ± 2  2 ±1 15 ± 5 Calendula flowers nd 3 ± 2 nd  7 ± 2 Onion bulb 5 ± 1 6 ± 2   2± 0.5  6 ± 2 Mushrooms 52 ± 8  6 ± 2 nd  55 ± 10 (Champignon) Cumin seed30 ± 6  15 ± 4  37 ± 7 120 ± 22 Apple fruit 4 ± 2 5 ± 2 10 ± 3  7 ± 2

Table 7. Total sRNA extracted from 100 g of plants and fungal tissueincubated with extraction media without tissue homogenization. The totalsRNA content of the liquid extract was determined using the Agilent RNA6000 Nano Kit through the Bioanalyzer instrument/Agilent Technologies)and data are expressed as ug/g of tissue±S.D.

This process allows to reduce costs and, after extraction of sRNAs, theplant biomass may be reused in the food chain and not becomingindustrial waste.

Further treatment of final sRNA extract:

The final extract may be concentrated through water removal(vacuum-drying) or filtration through size exclusion membranes (mustretain 13 KDa molecules).

-   -   1. RNAse

In order to improve the biological efficacy of the concentrated sRNAextracts, a method for reducing the possibility of off-targetsequence-dependent or RISC-mediated interference and/or pro-inflammatoryactivity of ssRNA molecules with length above 40 nt. was developed.Concentrated sRNA extracts were incubated with RNAse A (0.02 μg/10 μlextract) in the presence of 0.4M NaCl for 30 minutes at RT. With thistreatment 60%-90% of sRNA are removed and the resulting extract washighly enriched with dsRNA molecular species

-   -   2. dsRNA Purification Via dsRNA Binding Motifs (dsRBMs)

In order to improve the biological efficacy of the concentrated sRNAextracts, a method for reducing the possibility of off-targetsequence-dependent or RISC-mediated interference and/or pro-inflammatoryactivity of ssRNA molecules with length above 40 nt. was developed. RNAbiding proteins (RBP) abund in nature and are able to selectively andreversibly bind to RNAs. This ability is provided by specific RNAbinding domains (RBDs) which exists in various forms. In particular, thedouble stranded RNA binding domain (dsRBM) is a 70-75 amino-acid domainwhich plays a critical role in RNA processing, RNA localization, RNAinterference, RNA editing, and translational repression. The dsRBMsinteracts along the RNA duplex via both α-helices and β1-β2 loop andmake contact with the sugar-phosphate backbone of the major groove andof one minor groove, which is mediated by the β1-β2 loop along with theN-terminus region of the alpha helix 2. This interaction is a uniqueadaptation for the shape of an RNA double helix as it involves2′-hydroxyls and phosphate oxygen. Despite the common structuralfeatures among dsRBMs, they exhibit distinct chemical frameworks, whichpermits specificity for a variety for RNA structures includingstem-loops, internal loops, bulges or helices containing mismatches. TheCarnation Italian ringspot virus (CIRV) 19 kDa protein (p19), whichcontains a basic aminoacid signature that favor RNA binding, acts as adimer and binds with high affinity (nM-pM range) and littlesequence-specificity to the minor groove of siRNA duplexes ofselectively 21-25 nt. When p19 siRNA Binding Protein is expressed inplants it suppresses RNA interference.

Treatment of concentrated sRNA extracts (10 ug) with 200U p19 siRNAbinding proteins (product number M0310S NewEngland BioLabs) using theprotocol indicated in the Company datasheet (MOS10) resulted in removalof 95% of sRNA and purification of 21-24 nt dsRNA. This method can alsobe applied to the dsRNA enriched extracts following RNAse treatment (seeabove).

1. A method for isolating a fraction enriched of small RNA moleculesfrom a fungal and/or plant sample comprising: a) adding a lysis bufferto a powder or homogenized sample of fungal and/or plant tissue or cellssample to obtain a lysate; or a′) incubating fungal and/or plant tissueor cells with a bicarbonate solution at a temperature of 50-100° C.,separate liquid from solid phase to obtain a liquid phase; and b) addingan alcohol solution to the lysate obtained is step a) or to the liquidphase obtained in step a′) to obtain a solution; c) loading the obtainedsolution to a solid support able to selectively bind small RNAmolecules; and d) eluting small RNA molecules from said solid support.2. (canceled)
 3. (canceled)
 4. The method according to claim 1, whereinthe ratio w/v between powder and lysis buffer in step a) is of 1:1 to1:4.
 5. (canceled)
 6. The method according to claim 1, wherein the lysisbuffer comprises: 50 mM Tris-HCl (pH 8.5), 200 mM NaCl, 10 mM EDTA and2% SDS (w/v).
 7. The method according to claim 1, wherein thebicarbonate solution comprises 5-100 mM NaHCO3 solution.
 8. (canceled)9. The method according to claim 1, wherein the temperature of thebicarbonate solution is of about 95, 100 or 60° C.
 10. The methodaccording to claim 1, wherein before step a′) fungal and/or plant tissueor cells is incubated with a diluted bicarbonate solution at 0-10° C.,wherein the diluted bicarbonate solution is a 5-10 mM NaHCO3 solution.11. The method according to claim 1, wherein the alcohol is isopropanol,ethanol or any alcohol able to reduce the activity of water andtherefore the solvation of sRNAs and promote their binding to the solidsupport. 12-19. (canceled)
 20. The method according to claim 1, furthercomprising: step e) selectively removing ssRNAs with RNAse treatmentfrom the eluted small RNA molecules, and/or step f) treating the elutedsmall RNA molecules with an agent which binds with high affinity tosiRNA duplexes of selectively 21-25 nt, to enrich the 21-24 bp dsRNAfraction.
 21. The method according to claim 1, wherein the small RNAmolecules include miRNA, siRNA, snRNA, snoRNA, and/or tRNA molecules,preferably the small RNA molecules consisting of at most 100nucleotides, preferably at most 70 nucleotides, or more preferably atmost 30 nucleotides.
 22. The method according to claim 21, wherein thesmall RNA molecules consist of between 21 and 24 nucleotides, morepreferably between 19 and 24 nucleotides.
 23. The method according toclaim 1, wherein said small RNA molecules are in the single strandedand/or double stranded configurations.
 24. The method according to claim1, wherein said small RNA molecules are characterized by the presence ofa phosphate group at the 5′ ends and/or a methyl group at the 3′ ends.25. The method according to claim 1, wherein the small RNA molecules aremiRNA, mature miRNA and/or siRNA molecules. 26-30. (canceled)